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Nucleic Acids Research, 1981, Vol. 9, No. 21 5587-5600
© 1981

MOLECULAR BIOLOGY

Fine mapping of secondary structures of fd phage DNA in the region of the replication origin

Chiao-Chain Huang and John E. Hearst*

Department of Chemistry, University of California at Berkeley Berkeley, CA 94720, USA

* To whom reprint requests should be addressed.

Received July 21, 1981. A synthetic heptaribonucleotide, GACCCCC, which is complementary to a unique site on fd bacteriophage DNA, primes DNA synthesis of fd by T4 bacteriophage DNA polymerase. The rate of the GACCCCC-primed DNA synthesis was not uniform as reflected by the appearance of discrete DNA fragments as replication intermediates on an alkaline agarose gel. After 10 minutes of synthesis a signficant fraction of the DNA product ran as a single band with a length of about 1960 nucleotides. We have isolated this DNA fragment, hybridized back to unlabeled fd DNA template, and mapped the Taq 1 restriction fragments by urea polyacrylamide gel electrophoresis. This fine mapping procedure has located two major pause sites at fd nucleotide positions 5575 and 5674. These sites reside in the stem of two very stable hairpin helices near the origin of DNA replication of fd. Models for the functional roles of these two hairpin helices are presented.


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